This invention relates to methods of stimulation of host defense mechanisms against pathological conditions in a host mammal by administration of interferon via the oromucosa. In particular, the invention is applicable to methods of treatment of autoimmune, mycobacterial, neurodegenerative, parasitic, and viral diseases.
Interferon-xcex1 (IFN-xcex1) is used widely for the treatment of a variety of disorders including leukemia, lymphoma, AIDS-related Kaposi""s sarcoma, and hepatitis. (Gutterman, J. U., Proc. Natl. Acad Sci. USA, 1994 91: 1198-1205). Interferon-xcex2 (IFN-xcex2) is licensed for clinical use in treatment of relapsing-remitting multiple sclerosis and of chronic infection with Hepatitis B virus. Interferon-xcex1 and Interferon-xcex2 are both type I interferons. Although a number of routes of administration, including intravenous, subcutaneous, intramuscular, topical, and intralesional injection, are commonly employed for the administration of type I interferons, the oral route has not been generally used, because interferons are proteins which are considered to be inactivated by proteolytic enzymes and which are not absorbed appreciably in their native form in the gastrointestinal tract. Indeed a number of studies have failed to detect interferons in the blood following oral administration (Cantell and Pyhxc3xa4la, J Gen. Virol., 1973 20: 97-104; Wills et al, J IFN Res., 1984 4: 399-409; Gilson et al, J IFN Res., 1985 5: 403-408).
It has been shown that inducers of interferon are able to protect mice against experimental infection with Plasmodium berghei malaria, and that this protection is much more effective against sporozoite-induced infection than against infection induced by blood forms of the parasite (Jahiel et al, Science, 1986 16: 1802; Nature, 1968 220: 710; Amer. J. Trop. Med Hyg., 1969 18: 823). Mice injected intraperitoneally or intravenously with interferon in pooled serum of Newcastle disease virus-infected mice were protected against sporozoite-induced Plasmodium berghei malaria. However, interferon from rabbit serum was ineffective. Protection was obtained when the interferon was injected during the pre-erythrocytic phase of parasite development (i.e. three hours before or up to about 40 hours after sporozoite inoculation [Jahiel et al, Nature 1970 227: 1350-1351]).
There have been a number of anecdotal reports of efficacy of low doses of interferon administered as a nasal spray or as an oral liquid formulation in the treatment of a variety of viral conditions, particularly influenza. However, in most of these reports the interferon preparations used were relatively crude. Placebo-controlled trials of high dose intranasal interferon for treatment of rhinovirus infection showed that the treatment was effective, but that there was a significant incidence of side-effects (Hayden et al, J Infect. Dis., 1983 148: 914-921; Douglas et al, New Engl. J. Med., 1986 314: 65-80; Hayden et al; New Engl. J. Med., 1986 314: 71-75).
More recently a series of patent specifications has described the use of low doses of orally administered interferon of heterologous species origin for the treatment of infectious rhinotracheitis (xe2x80x9cshipping feverxe2x80x9d) in cattle, and of feline leukemia, and also treatment of other conditions, for enhancement of efficiency of vaccines; for improving the efficiency of food utilization; and for prevention of bovine theileriosis. See U.S. Pat. No. 4,462,985, Australian Patent No. 608519, Australian Patent No. 583332 and U.S. Pat. No. 5,215,741 respectively. In these specifications, the interferon used was human interferon xcex1 prepared by the method of Cantell, administered in phosphate buffered saline, at a dose of 0.01 to 5 IU per pound body weight. While these specifications suggest that such low doses of interferon administered to the oropharyngeal mucosa, preferably in a form adapted for prolonged contact with the oral mucosa, may be efficacious for treatment of a wide variety of conditions, the experimental evidence for conditions other than shipping fever, feline leukemia, canine parvovirus and theileriosis is largely anecdotal.
More recent studies on the effects of very low doses of interferon administered by the oral or oropharyngeal mucosa have been reviewed (Bocci, Clin. Pharmacokinet., 1991 21: 411-417; Critic. Rev. Therap. Drug Carrier Systems, 1992 9: 91-133; Cummins and Georgiades, Archivum Immun. Therap. Exp., 1993 41: 169-172). It has been proposed that this type of treatment is particularly useful for treatment of HIV infection, and can at least improve quality of life in AIDS patients (Kaiser et al, AIDS, 1992 6: 563-569; Koech et al, Mol. Biol. Ther., 1990 2: 91-95). However, other reports indicate that such treatments provide no clinical benefit. A Phase I study of use of oral lozenges containing low doses of interferon for treatment of hepatitis B has also been reported (Zielinska et al, Archiv. Immunol Therap. Exp., 1993 41: 241-252).
This invention provides a method for stimulating host defense mechanisms in a mammal via the oromucosal administration of an interferon. In one aspect, the invention may be considered as a method of stimulating the immune response in a mammal by administering to the mammal an immunostimulating amount of an interferon via oromucosal contact.
This invention provides a method for treating autoimmune, mycobacterial, neurodegenerative, parasitic, and viral diseases in a mammal via administering to the mammal a therapeutically effective amount of an interferon via oromucosal contact. The amount of interferon administered is less than an amount which induces a pathological response when administered parenterally. This invention provides a method for treating autoimmune diseases such as arthritis, diabetes, lupus, and multiple sclerosis, mycobacterial diseases such as leprosy and tuberculosis, neurodegenerative disorders such as encephalitis and Creutzfeldt-Jakob syndrome, parasitic diseases such as malaria, and viral diseases such as cervical cancer, genital herpes, hepatitis B and C, HIV, HPV, and HSV-1 and 2.
The oromucosal administration may involve administering an effective dose of interferon in a single dose or the effective dose may be administered in a plurality of smaller doses over a period of time sufficient to elicit host defense stimulation equivalent to that of a single dose. Likewise, the effective dose of interferon may be administered continuously over a period of time sufficient to elicit host defense stimulation equivalent to that of a single dose.
The method may be practiced by administering from about 1500 IU, preferably from about 5000 IU, to about 20xc3x97106 IU of interferon per day, more preferably from about 1xc3x97104 IU to about 20xc3x97106 IU of interferon per day, most preferably from about 1xc3x97104 to about 1xc3x97106 IU of interferon per day, provided that the chosen dose does not induce a pathological response when administered parenterally, or is less than a dose which would induce a pathological response when administered parenterally. These dose ranges generally refer to homologous interferon a in man. A physician treating a patient with a particular interferon will be able to readily identify the suitable therapeutic dose range according to the invention.
In another embodiment, the invention provides a pharmaceutical composition for oromucosal administration comprising a therapeutically effective amount of at least one interferon. The composition may be provided as a solution, tablet, lozenge, gel, syrup, paste, or controlled release oromucosal delivery system. Optionally, the composition may contain buffers, stabilizers, thickening agents, absorption, and viscosity enhancers, and the like.
In one embodiment, the pharmaceutical composition is provided in unit dosage form having from about 1500 IU, preferably from about 5000 IU, to about 20xc3x97106 IU of interferon, more preferably from about 1xc3x97104 IU to about 20xc3x97106 IU of interferon, most preferably from about 1xc3x97104 to about 1xc3x97106 IU of interferon.
The method may be practiced either as the sole therapeutic approach, or as an adjunct to other therapy, or with other cytokines, such as interleukin-2, 12, or 15, or with IFN-inducers.
The method is conducted using a Type I or II interferon, selected from xcex1, xcex2, xcex3, xcfx89, and consensus interferons, most preferably with a recombinant IFN-xcex1.
The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications referred to herein are expressly incorporated by reference.
Definitions
As used herein, xe2x80x9cinterferonxe2x80x9d refers to a Type I or Type II interferon, including those commonly designated as xcex1, xcex2, xcex3, and xcfx89, and mixtures thereof, including the consensus sequence. Interferons are available from a wide variety of commercial sources and are approved for the treatment of numerous indications. The interferon may be from natural sources, but is preferably a recombinant product. For the purposes of the invention, the term xe2x80x9cinterferonxe2x80x9d also includes polypeptides or their fragments which have interferon activity, and chimeric or mutant forms of interferon in which sequence modifications have been introduced, for example to enhance stability, without affecting the nature of their biological activity, such as disclosed in U.S. Pat. Nos. 5,582,824, 5,593,667, and U.S. Pat. No. 5,594,107 among others.
While it is to be clearly understood that the invention is applicable to any pathological condition in which stimulation of host defense mechanisms or immunostimualtion is beneficial, preferably the pathological condition is an autoimmune disease or a viral or parasitic infection. It will also be understood that for the purposes of the invention the interferon may be used either alone or in conjunction with another agent or treatment.
Optionally the interferon may be administered concurrently with an inducer of interferon synthesis and release. The inducer may be administered together with the interferon, or may be administered separately. A variety of inducers of interferon is known, for example polynucleotides such as poly I:C; preferably a low molecular weight, orally administrable interferon inducer is used. Suitable inducers are known in the art, for example, Tilorone (U.S. Pat. No 3,592,819; Albrecht et al, J. Med. Chem. 1974 17: 1150-1156) and the quinolone derivative Imiquimod (Savage et al; Brit. J. Cancer, 1996 74: 1482-1486).
The methods and compositions of the invention may optionally be used in conjunction with one or more other treatments for the specific condition, and the attending physician or veterinarian will readily be able to select such other treatment as may be appropriate in the circumstances.
The viral condition may be an acute or fulninant infection, such as rhinovirus, influenza, herpes varicella, herpes zoster, dengue fever, or viral encephalitis including but not limited to measles virus encephalitis, Murray Valley encephalitis, Japanese B encephalitis, tick-borne encephalitis and Herpes encephalitis; haemorrhagic fevers such as Ebola virus, Marburg virus, Lassa fever; Hanta virus infections, and other viral infections thought to be transmitted from animals to humans, such as equine morbillivirus. In many of these conditions there is no treatment and/or vaccine presently available, and supportive treatments may be inadequate. Alternatively the viral condition may be the result of chronic infection, such as hepatitis B, hepatitis C, hepatitis D or other forms of viral hepatitis, and CMV, HIV, HPV, and HSV I and II infection. Hepatitis B and hepatitis C are both currently treated with parenteral interferon; long-term interferon treatment in HIV infection which has progressed to AIDS is under clinical trial.
In a second embodiment, the disease to be treated is malaria, and again a Type I or II interferon is administered as described above. The causative organism of the malaria may be Plasmodium malariae, Plasmodium vivax, Plasmodiumfalciparum or Plasmodium ovale. It is particularly contemplated that the method of the invention will protect against progression of malaria to the cerebral form.
In a third embodiment, the invention provides a method of treatment of autoimmune disorders such as HIV, rheumatoid arthritis, and multiple sclerosis, whether of the relapsing-remitting or the chronic progressive type, or immunodeficiencies such as AIDS, comprising the step of administering an interferon as described above.
Again the method and dosage form of the invention may be used in conjunction with other treatments. For example, for herpes virus infection acyclovir or ganciclovir may be used. For HIV infection azidothymidine (zidovudine) or one or more other HIV reverse transcriptase inhibitors, and/or HIV protease inhibitors may be used.
In the preparation of the pharmaceutical compositions of this invention, a variety of vehicles and excipients for IFN may be used, as will be apparent to the skilled artisan. Representative formulation technology is taught in, inter alia, Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa, 1995, and its predecessor editions. The IFN formulation may comprise stability enhancers, such as glycine or alanine, as described in U.S. Pat. No. 4,496,537, and/or one or more carriers, such as a carrier protein. For example, for treatment of humans pharmaceutical grade human serum albumin, optionally together with phosphate-buffered saline as diluent, is commonly used. Where the excipient for IFN is human serum albumin, the human serum albumin may be derived from human serum, or may be of recombinant origin. Normally when serum albumin is used it will be of homologous origin.
The IFN may be administered by any means which provides contact of the IFN with the oromucosal cavity of the recipient. Thus it will be clearly understood that the invention is not limited to any particular type of formulation. The present specification describes administration of IFN deep into the oromucosal cavity; this may be achieved with liquids, solids, or aerosols, as well as nasal drops or sprays. Thus the invention includes, but is not limited to, liquid, spray, syrup, lozenges, buccal tablets, and nebuliser formulations. A person skilled in the art will recognize that for aerosol or nebuliser formulations the particle size of the preparation may be important, and will be aware of suitable methods by which particle size may be modified.
In one aspect, the interferon is administered in a single dose. Alternatively, the interferon is administered in a plurality of lower doses, distributed over time, so that the net effect is equivalent to the administration of the single higher dose. One approach to this delivery mode is via the provision of a sustained or controlled release device adhered to or implanted in the oromucosal cavity and designed to release interferon over time in an amount equivalent to a single high dose.
Representative formulations of interferon for oromucosal use include the following (all % are w/w):
Tablet: Dextrose BP 45%; gelatin BP 30%; wheat starch BP 11%; carmellose sodium BP 5%; egg albumin BPC 4%; leucine USP 3%; propylene glycol BP 2%; and 106 IU IFN-xcex12. The tablet may be used as is and allowed to slowly dissolve in the mouth or may be dissolved in water and held in the mouth as needed.
An interferon paste may be prepared, as described in U.S. Pat. No. 4,675,184, from glycerin 45%, sodium CMC 2%, citrate buffer (pH 4.5) 25%, distilled water to 100%, and 106 IU IFN-xcex12. The interferon paste may be adhered to the buccal mucosa.
Likewise, a gargle or a syrup may be prepared by adding the desired amount of interferon to a commercially available mouthwash or cough syrup formulation.
Within the specific dose ranges referred to above, the optimal treatment in any individual case will depend on the nature of the condition concerned, the stage of disease, previous therapy, other continuing therapy, the general state of health of the mammal, the sensitivity of the subject to interferon, etc., and therefore will be at the physician""s or veterinarian""s discretion, bearing in mind all these circumstances. The length of treatment will of course vary with the condition being treated, for example, treatment of an acute infection, such as Ebola virus, would be expected to involve a different course of treatment than treatment of a chronic condition, such as hepatitis.
The effective dose disclosed herein is one which does not generate a pathological response in the mammal when administered parenterally. A pathological response may be acute, chronic, or cumulative, and may be manifested by changes in blood chemistry, such as leukopenia, bone marrow depression, or other histological parameters. As used herein, a pathological response includes adverse side effects, such as fever, malaise, or flu-like symptoms, vascular reactions, such as phlebitis, and local inflammatory reactions at the site of injection. Such responses will vary considerably among the patient population in view of individual variations in sensitivity to interferon. A simple test for identifying an acceptable low dose of interferon for oromucosal therapy is to inject the patient with the putative acceptable dose, based upon considerations of age, weight, indication, progression, etc. and ascertain if the injection produces a pathological response as defined herein, with local irritation at the site of injection being the most readily ascertainable criterion. If no adverse response is noted, then the same dose may be administered oromucosally. If there is an undesirable response, then the process is repeated at a lower dose, until a non-pathological dose is identified.
For many patients, it is expected that oromucosal doses will be approximately the same as those known to be well tolerated and effective in existing approved parenteral protocols. Therefore, for purposes of specificity, an acceptable low dose of interferon may be from about 1500 IU, preferably from about 5000 IU, to about 20xc3x97106 IU of interferon. More preferably the dose is from about 1xc3x97104 IU to about 20xc3x97106 IU of interferon, most preferably from about 1xc3x97104 IU to about 1xc3x97106 IU of interferon, provided that the dose is one which does not induce a pathological response when administered parenterally. In one embodiment, the total dose may be administered in multiple lower doses over time, or even may be delivered continuously or in a pulsatile manner from a controlled release device adhered to or implanted in the oromucosa.
Mouse IFN-xcex1/xcex2 (Mu IFN-xcex1/xcex2) was prepared from cultures of C243-3 cells induced with Newcastle disease virus (NDV) and purified as described previously (Tovey et al, Proc. Soc. Exap. Biol. and Med., 1974 146: 809-815). The preparation used in this study had a titer of 4xc3x97106 International Units (IU)/ml and a specific activity of 5xc3x97107 IU/mg protein as assayed on mouse 929 cells challenged with vesicular stomatitis virus (VSV) as described previously (Tovey et al, Proc. Soc. Exp. Biol. and Med., 1974 146: 809-815). The preparation was standardized against the international reference preparation of murine IFN-xcex1/xcex2 of the National Institutes of Health (NIH) (G-002-9004-5411).
Human IFN-xcex1 1-8
Recombinant human IFN-xcex1 1-8 (Hu IFNxcex1 1-8; BDBB lot no. CGP 35269-1, Ciba-Geigy, Basel, Switzerland) was prepared and purified as described previously (Meister et al, J Gen. Virol., 1986 67: 1633-1643). The preparation used in this study had a titer of 70xc3x97106 IU/ml on homologous human WISH cells challenged with VSV as described previously (Tovey et al, Nature, 1977 267: 455-457), and a titer on heterologous mouse L929 cells of 1xc3x9710 IU/ml. The preparation was standardized against both the NIH human IFN-xcex1 international reference preparation (G-023-901-527) and the NIH murine IFN-xcex1/xcex2 standard (G-002-9004-5411). The specific activity of the IFN preparation was 2xc3x97108 IU/mg protein.
Recombinant murine interferon-xcex1 was purchased from Life Technologies Inc. The preparation used in this study (lot no. HKK404) had a titer of 6xc3x97106 IU/ml and a specific activity of 6xc3x97108 IU/mg protein as assayed on mouse L929 cells challenged with VSV (Tovey et al, Proc. Soc. Exp. Biol. Med., 1974, 146:406-415).
Recombinant murine interferon xcex2 was purchased from R and D Systems Inc. The preparation used in this study (lot no. 1976-01S) had a titer of 3.2xc3x97104 IU/ml and a specific activity of 8xc3x97106 IU/mg protein as assayed on mouse L929 cells challenged with VSV (Tovey et al, Proc. Soc. Exp. Biol. Med., 1974, 146:406-415).
Recombinant murine interferon xcex3 was purchased from R and D Systems Inc. The preparation used in this study (2580-03SA) had a titer of 2xc3x97105 IV/ml and a specific activity of 1xc3x97107 IU/mg protein as assayed on mouse L929 cells challenged with VSV (Tovey et al, Proc. Soc. Exp. Biol. Med., 1974, 146:406-415).
All the interferon preparations were titrated simultaneously in the same assay and standardized against the international reference preparation of murine interferon xcex1/xcex2 of the US National Institutes of Health (G-002-9004-5411).
Interferon preparations were diluted in phosphate buffered saline (PBS) containing bovine serum albumin (BSA) or a proprietary excipient. Bovine serum albumin fraction V (RIA grade; immunoglobulin free; Cat. no. A7888; Sigma; USA) was dissolved at a final concentration of 100 xcexcg/ml in PBS (pH 7.4) and sterilized by filtration (0.2 xcexcl, Millex-GV, Millipore, USA). The proprietary excipient used was as follows, supplied in the form of tablets (Ferimmune(trademark), Pharma Pacific):
A single tablet was dissolved in 1.5 ml phosphate buffered saline, centrifuged at 16,000 g for 15 m, and then sterile filtered (0.2 xcexc, Millex-GV, Millipore, USA), and stored at 4xc2x0 C. prior to use. Excipient was prepared daily prior to use.
Preliminary experiments showed that the application of 5 xcexcl of crystal violet to each nostril of a normal adult mouse using a P20 Eppendorf micropipette resulted in an almost immediate distribution of the dye over the whole surface of the oropharyngeal cavity. Staining of the oropharyngeal cavity was still apparent some 30 minutes after application of the dye. Essentially similar results were obtained using 125I-labelled recombinant human IFN-xcex1 1-8 applied in the same manner. This method of administration was therefore used in all subsequent experiments.
For the purposes of the animal experiments described in this specification, it will be clearly understood that the expression xe2x80x9cintranasal/oralxe2x80x9d or xe2x80x9cintranasal plus oralxe2x80x9d or xe2x80x9cin/orxe2x80x9d or xe2x80x9coromucosalxe2x80x9d or xe2x80x9coropharyngealxe2x80x9d with reference to the route of administration of IFN is to be taken to mean administration of the IFN preparation deep into the nasal cavity so that it is rapidly distributed into the oropharyngeal cavity, i.e. the mouth and throat of the recipient mammal, so as to make contact with the mucosa lining this cavity.
Batch: Lot no. 095001
Expiration Date: December 1997
Preparation: EMCV strain JH was propagated on mouse L929 cells using methods described previously (Gresser I. Bourali C, Thomas M T, Falcoff E. Effect of repeated inoculation of interferon preparations on infection of mice with Encephalomyocarditis virus. Proc Soc Exp Biol Med 1968 February; 127:491-6)
Characterization: The virus stock used in this study had a titer of 5xc3x97108.62TCID50 on mouse L929 cells.
Storage: Stock EMCV was stored at xe2x88x9270xc2x0 C. A power cut on day 1 of the Virus Titration necessitated transfer temporarily to back-up storage at approximately the same temperatures. The material remained frozen at all times. On day +8 of the Virus Titration the xe2x88x9270xc2x0 C. freezer increased in temperature to xe2x88x9260xc2x0 C. Diluted EMCV was prepared immediately before use and was kept on ice or in the animal room refrigerator until use.
The mice used in this study were obtained from a specific pathogen-free colony (IFFA CREDO, France). They were housed in a specific pathogen-free animal facility at the Institut Federatif CNRS at Villejuif according to EEC standards.
Interferon was assayed according to a conventional method. Briefly, samples (20 xcexcl) were diluted in 80 xcexcl of Eagle""s Minimal Essential Medium (MEM) (Gibco, France) containing 2% heat-inactivated Fetal Calf Serum (FCS) (Gibco, France) and added to each well of a microtiter plate (Falcon, cat. no. 3072) using a multichannel micro-pipette (Finnpipette, Labsystem, 50-300 xcexcl). WISH or L929 cells (2xc3x97104 cells/well) were added in 100 xcexcl of MEM containing 2% FCS and incubated overnight at 37xc2x0 C. in an atmosphere of 5% CO2 in air (Forma 3029 CO2 incubator). The cells were then examined for any signs of toxicity using an Olympus IM GLDW inverted microscope equipped with a 10xc3x97 objective. Samples which did not exhibit detectable toxicity were then subjected to serial two-fold dilutions starting from an initial 1:10 dilution in a total volume of 200 xcexcl of Eagle""s MEM containing 2% FCS, by carrying forward 100 xcexcl of diluted material with a multichannel micropipette, in a microplate containing 100 xcexcl per well of fresh Eagle""s MEM containing 2% FCS. Appropriate serial two-fold dilutions of the NIH human IFN-xcex1 reference standard (G-023-901-527) or the NIH Mu IFN-xcex1/xcex2 reference standard (G002-9004-5411) were also prepared. WISH or L929 cells (2xc3x97104 cells/well) in 100 xcexcl of Eagle""s MEM containing 2% FCS were then added to each plate where appropriate and incubated overnight at 37xc2x0 C. in an atmosphere of 5% CO2 in air. The cell monolayers were then checked for any signs of toxicity and in the absence of any apparent toxicity, the culture was aspirated and replaced with 200 xcexcl of Eagle""s MEM containing 2% FCS containing 100 TCID50 of VSV (2xc3x9710xe2x88x924 VSV23 for WISH cells, or 10xe2x88x925 VSV23 for L929 cells). The plates were then incubated overnight at 37xc2x0 C. in an atmosphere of 5% CO2 in air. The cell monolayers were then examined for specific viral cytopathic effect using an Olympus IM ULWD inverted microscope. Interferon titers were determined from the reciprocal of the dilution which gave 50% protection against specific viral cytopathic effect, and are expressed in international reference units/ml (IU/ml).